Apoptosis or programmed cell death is a form of active cell death essential during the development and maintenance of tissue homeostasis and for the elimination of damaged cells (Saunders, Science 154:604 (1996); Kerr et al, Br. J. Can. 26:239 (1972)). Apoptotic cell death is also involved in a wide range of pathological conditions including cancer and immunological and neurodegenerative diseases (McDonnell, Mol. Carcinogen 8:209 (1993); Camilleri-Broert et al, Blood 86:432 (1995); Migheli et al, Neuroreport 15:1906 (1994)). Regulation of the apoptotic process involves the activation of intracellular proteins, including proteins of the Bcl-2 family. Bcl-2 was first identified as an inhibitor of apoptosis following its identification in follicular B-cell lymphoma (Tsujimoto et al, Science 228:1440 (1985); Vaux et al, Nature 335:440 (1988)). The members of the Bcl-2 family exhibit either anti-apoptopic (Bcl-2, Bcl-xl, Mcl1, Ced9) or pro-apoptotic (Bax, Bak, Bad, Bcl-Xs) properties (Korsmeyer, Trends 11:101 (1995)). It has been proposed that the activity of the proteins is regulated through the formation of homo- and hetero-complexes (Borner et al, Biochem. Cell Biol. 72:463 (1994); Farrow et al, Curr. Opin. Gen. Dev. 6:45 (1996)). One model, the rheostat model, proposes that the homo-complex of Bax induces downstream death activators and the hetero-complex with Bcl-2 inactivates Bax by preventing homo-complex formation (Oltavai et al, Cell 74:609 (1993)). The equilibrium between homo- and hetero-complexes can be regulated either at the transcriptional level or through post-translational modifications, in particular, phosphorylation. Bcl-2 phosphorylation has been shown to modulate its ability to prevent cell death (Haldar et al, Proc. Natl. Acad. Sci U.S.A. 92:4507 (1995); Chen et al, J. Biol. Chem. 271:2376 (1996)). Bad, a pro-apoptotic member of the family, has also been shown to be phosphorylated (Zha et al, Cell 87:619 (1996)).
The proteins contain three highly conserved regions designated BH1, BH2 and BH3. The BH3 domain is essential for the death promoting activity of the pro-apoptotic proteins (Hunter et al, J. Biol. Chem. 271:8521 (1996)) whereas BH1 and BH2 are required for the formation of protein complexes (Borner et al, Biochem. Cell Biol. 72:463 (1994); Farrow et al, Curr. Opin. Gen. Dev. 6:45 (1996)). The proteins have the potential of being membrane attached and contain a predicted membrane spanning domain at the COOH-terminus which gives the proteins a low solubility once extracted from their natural environment. Bcl-2 has been shown to be predominantly localized to the mitochondrial outer membrane, but the protein also is found in the endoplasmic reticulum and the nuclear membrane (Krajewski et al, Cancer Res. 53:4701 (1993)). Recently, Bax and Bcl-x.sub.L were shown to translocate from the cytosolic fraction to the membrane fraction when apoptosis was induced in murine thymocytes (Hsu et al, Proc. Natl. Acad. Sci. U.S.A. 94:3668 (1997)). In HeLa cells, overexpressed Bax is localized predominantly to the mitochondrial and the endoplasmic reticulum membranes.
Recently, the X-ray and NMR structures of Bcl-x.sub.L were solved (Muchmore et al, Nature 381:335 (1996)). The .alpha. helical structures present in bacteria toxins, colicins and diphteria toxin, which are believed to be associated with their pore-forming activity, are apparently conserved in Bcl-X.sub.L (where they are coded for by the amino acids linking the BH1 and BH2 domains) thereby suggesting that Bcl-X.sub.L may have pore-forming activity. Indeed, Bcl-x.sub.L was subsequently shown to form ion channels in synthetic lipid membranes (Minn et al, Nature 385:353 (1997)).
Although Bcl-x.sub.L and Bax belong to the same protein family they have opposite activities; Bcl-x.sub.L anti-apoptotic activity and Bax pro-apoptotic activity. Indeed, the amino acid sequence identity between Bcl-x.sub.L and Bax is only 26% with the highest homologies in the BH1-3 domains. Moreover, the sequence between the BH1 and BH2 domains coding for the putative pore-forming .alpha. helices in Bcl-x.sub.L is not at all conserved in Bax. The present invention results from the finding that, despite the opposite activities, relatively low sequence homology and lack of conservation of the region between BH1 and BH2, Bax, like Bcl-x.sub.L, has channel-forming activity.